Probably the greatest challenge presently facing comprehensive proteome analysis is related to the large variation of protein relative abundances (>6 orders of magnitude), having potential biological significance in mammalian systems. Additionally, limited sample amounts ranging from 1,000-100,000 cells are available in mammalian proteomics, corresponding to a total protein content of 0.1-10 micrograms. For example, the use of laser capture microdissection techniques yields tissue volumes in cubic micrometers and sample sizes in the sub-microgram range. Furthermore, the heterogeneous nature of cells and tissues also contributes to the requirement for analyzing limited subpopulations. The R43 Phase of this project aims to develop and demonstrate a transient capillary isotachophoresis/zone electrophoresis (CITP/CZE)-based multidimensional separation platform, capable of providing significant analyte concentration through electrokinetic stacking (400-5000 fold) and extremely high resolving power toward peptide and protein mixtures. Most importantly, the final concentration of the analyte achieved in CITP is largely proportional to the molarity of the leading buffer. Thus, the result of the CITP process is that major components may be diluted, but trace compounds are concentrated. Such selective enhancement toward low abundance proteins can drastically reduce the range of relative protein abundances in complex proteomes, and greatly improve proteome coverage using the proposed CITP/CZE-based multidimensional separation technology. In comparison with the displacement chromatography, the only reported approach in the literature to this point for the selective enrichment of low abundance peptides, transient CITP/CZE offers the benefits of speed, straightforward manipulation/switching between the stacking (CITP) and separation (CZE) modes, and no need for column regeneration, including the removal of bound displacers. Research efforts in the R43 Phase will address early implementation, optimization, and demonstration of the proposed CITP/CZE-based multidimensional separation platform for proteomic analysis of yeast tryptic peptides. The results of R43 Phase studies will allow validation of technology, and provide information necessary to guide the improvement, design, and completion of an automated, high throughput, robust, sensitive, and ultrahigh resolution proteome instrument in the R44 Phase studies.